The goal of this protocol is to present an efficient method for identifying neurons patched during electrophysiological recordings using biocytin and immunostaining for recovery of morphology prior to re-staining with specific antibodies to determine neurochemical identity. So this method describes an important step needed to study the diversity in neuronal structure and function. This is essential to understand their role in brain networks.
The main advantage of using a sequential processing techniques, is that it eliminates the data loss because of inability to recover morphology. So this helps also guide the neurochemical markers to be tested in these cells. Although this method is described for morphological recovery of cells in thick sections, it can also be applied to thin sections and on previously stained and mounted samples.
Generally neutrals new to this method may have difficultly in resealing the soma while detaching from the cell, and may fail to allow adequate time for successful fills. Visual demonstration of this method is critical as specific techniques are required for effective biocytin filling of cells in order to recover the complete morphology. To begin this procedure, in voltage clamp configuration apply small brief voltage steps using the membrane test function in bath mode while approaching the cell.
Patch onto the cell by applying suction. Then, hold the cell at negative 70 milivolts. Note the development of a gigaohm seal.
Compensate for capacitive transients and then apply controlled suction with a syringe or by mouth to achieve whole cell configuration. Estimate whole cell capacitance and series resistance, and stop the membrane test pulses. Series resistance of less than 10 megaohm is optimal.
This will ensure the establishment of whole cell recording conditions to allow for biocytin filling. Upon completion of the physiological recordings, re-establish the patch clamp configuration by slowly moving the recording pipette in small steps, alternating upward and outward in voltage clamp mode. The most critical step in accomplishing morphological recovery of cells, is to ensure the re-sealing of the neuronal soma while detaching the recording pipette from the cell.
Using the membrane test function, visualize the loss of capacitive transients and the collapse of current responses to a straight line, which indicates the re-sealing of the cell and the establishment of an outside out patch at the pipette tip. In the meantime, holding the cell at a depolarized potential will facilitate the resealing process. After detaching the pipette from the cell, retain the slice in the recording chamber for three to five minutes to ensure the dye transporting to the distal dendritic and axonal processes.
Then, transfer the slice to a 24-well plate containing 4%PFA for fixation. 24 to 48 hours after PFA fixation, transfer all the fixed slices to zero point one molar PBS for storage prior to immunostaining. In this procedure, wash the tissues three times for 10 minutes each in zero point one molar PBS.
Next, block the tissues with 10 percent blocking serum for one hour at room temperature. Subsequently, incubate the sections in primary antibodies CB1R at room temperature overnight to identify cannabinoid receptor type 1 expression. Then, wash the brain sections three times for 10 minutes each in 0.1 molar PBS.
Streptavidin and secondary antibody to first primary antibody are both added to the well plate and kept overnight at four degrees celsius. Subsequently, incubate the sections in streptavidin red dye conjugate at four degrees celsius overnight in the dark to reveal the biocytin. After that, wash the brain sections three times for 10 minutes each in 0.1 molar PBS.
To protect the florescence and to facilitate restaining in the future, mount the sections at an equally spaced mounting medium. Enseal the edges of the cover slips with clear nail polish. In this step, strip the nail polish off of the glass slide with a cotton swab of acetone.
Remove the cover slip carefully using fine tip forceps and put a few droplets of 0.1 molar PBS on the sections. It is critical not to damage the process tissue while unmounting the tissue from the aqueous mounting medium. Carefully remove the sections from the slide using a fine paintbrush and wash them in 0.1 molar PBS for 24 to 48 hours on a shaker, and kept away from light in four degrees Celsius.
To ensure complete removal of the mounting medium, wash the sections one to two times on the following day in 0.1 molar PBS for 10 minutes each time. Then, incubate the sections with a second primary antibody cholecystokinin for one day. Wash the brain sections three times for 10 minutes each in 0.1 molar PBS.
Subsequently, stain the sections with a fluorophore conjugated secondary antibody goat anti-mouse. Making sure that the excitation emission spectra of the secondary antibody do not overlap with streptavidin and prior immunofluorescent stains. Afterward, mount the sections in aqueous mounting medium.
Enseal the edges of the cover slip with clear nail polish. Following successful biocytin fill and the first immunostaining process, the neuronal structure can be visualized and imaged as shown in the confocal image obtained at 60x magnification. The section was also immunostained for CB1R along with biocytin, and the CB1R immunostaining in the biocytin expressing neuronal soma is indicated by the arrowhead.
The same section was processed for CCK immuno-reactivity after one week, and the image shows successful recovery of CCK immuno labeling in several neurons indicated by the asterisk. Note that the biocytin labeled cell indicated by the arrowhead did not express CCK. A merged image of panels A, B, and C show that all three labels are visualized robustly, and the overlay between biocytin and CB1R is evident.
The image shows that the second immunostaining process does not compromise the signal from the prior staining process. Morphology of the biocytin filled cell was obtained from the confocal images prior to this CCK immunostaining. Crucially, in addition to the soma and dendrite, axons can be visualized, and co-labeling of CB1R and biocytin labeled axonal processes is observed.
While attempting this procedure, it's important to target healthy cells located below the slice surface to avoid neurons with severed processes. And that could compromise the recover of the morphology. Once mastered, morphological recovery of biocytin filled cells can be reliably obtained during the patch clamp recording.
Successful implementation of serial immunostaining technique will improve efficiency of data recovery and facilitate neuronal characterization during standard patch clamp recordings. After watching this video, you should have a good understanding on how to effectively use biocytin filled cells to recover neuronal morphology and to determine neurochemical markers.
